Heterosexual HIV-1 transmission to women accounts for the majority of new infections worldwide, and effective prevention modalities will likely have to interdict virus or virally-infected cells prior to HIV-1 dissemination to secondary lymphoid compartments. However, the biological and molecular basis of early virus-host interactions that lead to the establishment of HIV-1 infection in the female genital tract mucosa remains poorly understood. Studies of transmitted/founder (T/F) viruses revealed a phenotype of weak macrophage tropism, and in primate model studies resting memory CD4+ T cells were the principal early target of SIV infection. One interpretation of these findings is that HIV-1 macrophage (Mf) tropism is not an important property for mucosal HIV-1 transmission, but emerges later in HIV-1 pathogenesis. Consequently, the potential role of tissue mucosal macrophages (mMfs) in male-to-female HIV-1 transmission has largely been discounted. In a recent report we now show that, through a previously unrecognized pathway, monocyte-derived macrophages (MDM) can be efficiently infected with R5 (T/F) but not X4 viruses as they specifically recognize and phagocytose infected CD4+ T lymphocytes (Baxter et al., 2014). If this mechanism was operative in vivo, it would have important implications for understanding HIV-1 transmission. Infected Mf can harbor, produce and release infectious HIV-1 without succumbing to cytopathic effects, and, thus, if resident mMf infection occurred (via such pathway) within the first few days, it could drive the expansion of virus beyond localized foci that, in NHP models, have been observed in the vaginal mucosa just days after exposure to SIV. We submit that our findings provide a compelling, potentially paradigm-shifting argument for interrogating the potential role of cervicovaginal mMfs in virus-host interactions in early infection and virus amplification in the female genital tract mucosa, and possibly gain new insights into selective R5 virus transmission. To test our HYPOTHESIS that T/F (R5) virus infection spreads from infected CD4+ T lymphocytes to cervical tissue Mfs, and early productive infection of Mf in the cervical mucosa augments virus replication and spread to surrounding mucosal cell targets, we will utilize complementary human cervical cell models of HIV-1 transmission and innovative virologic tools. We will investigate two SPECIFIC AIMS: 1. Elucidate the susceptibility of cervical Mf and CD4+ T cells, isolated from mucosal tissues, to T/F and X4 strains of HIV-1 by cell-free and cell-cell-mediated pathways of virus infection; and 2. Elucidate the susceptibility of Mf in situ, in cervical tissue explants, to T/F strains of HIV-1by cell-free and cell-cell-mediated infection pathways, and their contribution to virus amplification and spread within tissue. This project is of potential high impact, as it addresses fundamental questions of HIV-1 transmission, and complements existing models of rectal and cervicovaginal SIV transmission. Our work will contribute new insights into HIV/SIV transmission highly relevant to the development of efficacious prevention strategies.